Co-targeting JAK1/STAT6/GAS6/TAM signaling improves chemotherapy efficacy in Ewing sarcoma

Ewing sarcoma is a pediatric bone and soft tissue tumor treated with chemotherapy, radiation, and surgery. Despite intensive multimodality therapy, ~50% patients eventually relapse and die of the disease due to chemoresistance. Here, using phospho-profiling, we find Ewing sarcoma cells treated with chemotherapeutic agents activate TAM (TYRO3, AXL, MERTK) kinases to augment Akt and ERK signaling facilitating chemoresistance. Mechanistically, chemotherapy-induced JAK1-SQ phosphorylation releases JAK1 pseudokinase domain inhibition allowing for JAK1 activation. This alternative JAK1 activation mechanism leads to STAT6 nuclear translocation triggering transcription and secretion of the TAM kinase ligand GAS6 with autocrine/paracrine consequences. Importantly, pharmacological inhibition of either JAK1 by filgotinib or TAM kinases by UNC2025 sensitizes Ewing sarcoma to chemotherapy in vitro and in vivo. Excitingly, the TAM kinase inhibitor MRX-2843 currently in human clinical trials to treat AML and advanced solid tumors, enhances chemotherapy efficacy to further suppress Ewing sarcoma tumor growth in vivo. Our findings reveal an Ewing sarcoma chemoresistance mechanism with an immediate translational value.

sensitive and presuming that these treatments at 24 hours are leading to chemoresistance is not well justified.
Technical comments: Figure 1: Explain the rationale for the concentrations used in Figure 1 (some seem quite high) as well as the timepoint for analysis at 24hrs.What happens if assess phospho-proteins at 12 hours or 48 hours?Would also be appropriate to measure timeline of drug treatment in the text.
What % of cells undergoing apoptosis at this timepoint?Did the authors assess any combinations tried to see if effects p-status of pathways and try to recapitulate the patient treatment regimen Were any upstream kinases, such as AXL, MET on the phosphoprofile?How come activation of these signaling components not noted?Please comment/address.Show tumor response?This still does not prove these are associated with driving resistance.Also, the authors should assess with multiple models and chemotherapies…not just irinotecan.For Fig 1m, they are biased towards looking at p-ERK and p-AKT.Should do phosphoarray analysis on these tumors?Other pathways that might be contributing to the "chemosresistance", which again needs to be more convincingly demonstrated.
Major issue (goes back to the conceptual point noted above) is that the authors use   Show that GAS6 is a direct target of STAT6?Or an indirect mechanism?Need to further prove or discuss.What are the effects of phosphorylation at these higher doses of chemo?IC50s are 10fold higher than used for WBs.At these higher concentrations (1 0-300uM)what other signaling pathways are being altered?This needs to be discussed as well.Fig 8 .In vivo studies would benefit from assessing the effects on more than 1 cell line model.
Reviewer #3: Remarks to the Author: Yu and colleagues present an interesting study on signaling pathways that contribute to chemoresistance in Ewing sarcoma and provide data that nominate potential effective combination strategies.The authors provide evidence that chemotherapy induces phosphorylation/activation of multiple signaling proteins, notably AKT-T308, in Ewing sarcoma cells.The authors suggest that this chemo-induced signaling is dependent on TAM receptor activation via upregulation of Gas6 (presumably autocrine activation).The authors show that Gas6 induction is STAT6-mediated and that STAT6 is activated by JAK1, which itself is stimulated as a result of chemotherapy.Therapeutic implications are highlighted through xenograft studies where inhibition of JAK1 or TAM receptors in combination with chemotherapy potently reduces Ewing xenograft growth.
There are multiple noteworthy results.1. Demonstration of a plausible pathway that induces TAM (Axl/MerTK) activation by chemotherapy; 2. Use of multiple Ewing cell lines increases rigor in the study; 3. Loss/Gain of function and pharmacologic inhibition of the TAM pathway results in consistent phenotypes in multiple cell lines, again increasing rigor; 3. Activation of JAK1 by chemo is a novel aspect of the study and the authors provide evidence to support this claim.
The work is original.There is limited published data on the contribution of Axl, MerTK, JAK1 and STAT6 individually in Ewing sarcoma.The concept that combination therapy is needed to improve outcomes in Ewing sarcoma is not novel.However, the strategy of looking for chemo-activated pathways in tumor cells and then targeting those pathways as a route to an effective combo is novel.
The methods are adequately described (except where noted below) and the study encompasses a wide breadth of techniques, results of which in general support the overall conclusions of the study.
Major Comments: 1.In general additional detail should be added to the figure legends and/or methods section where appropriate.For example, Figure 1B, the vendor and cat # for the membrane used to probe phospho-proteins should be provided and the conditions used to stimulate cells before lysate production should be clarified (e.g., how long were cells stimulated with .A key detail that should be provided in the figure legends is time frame of stimulation or assay.
2. Was AKT-pS473 included in the membrane shown in Fig 1B ?The authors only show data with AKT-pT308, why?If AKT-s473 is not phosphorylated is the AKT pathway relevant to chemoresistance?
3. The activation of JAK1 by chemotherapy needs to be clarified.The text presents the concept that DNA damage response kinases (ATM, ATR, DNAPK) can phosphorylate JAK1 at S571 and this is repeated in the discussion.However, the authors do not mention that a SQ sequence is indicative of an ATM/ATR substrate site.It will be confusing for readers to just jump to JAK1-pSQ terminology.As written there are sections of the results that read as if JAK1 activation occurs in the absence of DNA damage response.Please clarify.
4. Does activation of JAK1 by chemo occur in the nucleus or cytoplasm? 5.The in vivo efficacy studies are promising.I strongly suggest/recommend repeating in vivo studies testing the best combo therapy vs chemo alone and control (limited groups) in mice with established tumors (>250 mm3) to drive higher impact.

Minor:
1.In Fig 2 the authors show that KD of AXL blocks chemo-induced AKT-pT308 but cabozantinib, a reasonably potent inhibitor of AXL, does not.This is somewhat confusing and should be discussed.Reviewer #4: Remarks to the Author: This manuscript reports very interesting studies on the understanding of the molecular basis of Ewing sarcoma chemoresistance.The authors provide strong data to suggest that chemotherapy induces JAK1 activation via DNA damaging kinases-mediated JAK1-SQ phosphorylation, which leads to the subsequent activation of STAT6, resulting in the transcriptional activation of GAS6.The secretion of GAS6 protein induces TAM kinase activation and then the increased activation of Akt and ERK in Ewing sarcoma cells.The authors propose that this newly identified JAK1/STAT6/GAS6/TAM/Akt-ERK pathway contributes to Ewing sarcoma chemoresistance.In support of their hypothesis, JAK1 inhibitor filgotinib or TAM kinase inhibitor UNC2025A sensitized Ewing sarcoma cells and tumors to chemotherapy in vitro as well as in animal tumor models.In addition, the TAM kinase inhibitor MRX-2843 currently in clinical trials to treat AML and advanced solid tumors showed synergistic effects with chemotherapy to inhibit Ewing sarcoma tumor growth.These results have potential translational value to overcome Ewing sarcoma chemoresistance.There are a few issues that the authors should address.1) Several previous studies have reported the involvement of STAT3 in the development of chemoresistance, and GAS6 is known to induce the activation of STAT3.The authors should provide experimental data to address if STAT3 plays any role in Ewing sarcoma chemoresistance.

I recommend
2) Have the authors tested if JAK1 becomes tyrosine phosphorylated/activated by chemo drugs in Ewing sarcoma cells?
3) Can the authors show the direct binding of STAT6 to the promoter of GAS6 gene by ChIP assays?4) Serine phosphorylation of STATs is important for STAT-mediated gene activation.Several kinases including ERKs are involved in STAT serine phosphorylation.Since chemotherapy was shown to induce ERK activation in Ewing sarcoma cells, does the chemotherapy-induced ERK activation have any effect on STAT serine phosphorylation, if so, does this phenomenon contribute to the chemoresistance in Ewing sarcoma?Response: We thank the reviewer for raising this great question and fully agree that we should also examine effects of inhibitor treatment alone.We have repeated these experiments and included inhibitor alone conditions.As shown in the revised Fig. 6f-6k, inhibitor alone didn't significantly affect Akt or ERK activation.

Reviewer #2
In this manuscript by Yu et al., the authors hypothesized that Ewing sarcoma cells acutely adapt to chemotherapy by altering specific signaling pathways prior to genomic changes and investigate the role of the JAK1-STAT6-GAS6-TAMK signaling network and its role in Ewing sarcoma chemosensitivity.The authors establish a chemotherapy-driven intrinsic mechanism of therapeutic resistance through the activation of JAK-STAT signaling, which then transcriptionally activates GAS6.GAS6 is then secreted and has an autocrine/paracrine effect on the tumors cells to activate the AKT and ERK pathways and direct chemoresistance.The mechanism and connections between the intrinsic activation to secretion of TAM activating ligand is well described as well as the step wise activation of the individual components of the signaling cascade.Also, they show that targeting components of this pathway have additive/synergistic effects in vivo.However, there are some concerns regarding the conceptual as well as technical aspects of the manuscript that need to be addressed.

Response:
We thank the reviewer for acknowledging the importance and therapeutic potential for our study.We also thank the reviewer for raising these great suggestions to further improve our manuscript.Please find our detailed point-to-point responses to each raised question below.

Conceptual issues:
1. Patients treated for Ewing sarcoma see a combination of chemotherapy, which includes simultaneous vincristine, doxorubicin and Cytoxan which is then alternated with Ifosfamide and Etoposide for upfront therapy.For relapse therapy it is unusual for just a single agent to be administered as well.A typical relapsed therapy includes vincristine, irinotecan and temozolomide (VIT), and both Irinotecan and TMZ are given on days 1-5.So how will tumor cells react to combo that simulates actual therapy.So while the authors investigate the effects of single agent therapy, the mechanisms of resistance are most likely different upon combination therapy.
Response: We thank the reviewer for raising this critical question and fully agree with the reviewer that initial therapy for localized Ewing sarcoma involves 5 drugs used in an alternating pattern, including doxorubicin and etoposide.However, in the context of recurrence, irinotecan (which is metabolized to the topoisomerase I inhibitor, SN-38) and temozolomide (TMZ) have been shown to be temporarily effective for a large fraction of patients (Casey et al. Pediatr Blood Cancer. 2009. 53: 1029-1034), and this combination has been used together with experimental agents (Federico et al., 2020. Eur J Cancer. 137: 204-213;Wang et al. 2022. BMC Cancer. 22: 349).
We fully agree with the reviewer that it is critical to test if combo-chemo behaves similar to single chemo in our signaling studies.We compared the combination of SN-38 with TMZ to each single treatment in three Ewing sarcoma cells at doses that neither of the treatment killed cells in a 24-hr period, and found that combination treatment further increased ERK-p42/p44 with minimal effects on Akt-pT308 compared with each single chemo-treatment in A673, MHH-ES-1 and TC-71 cells (revised Supplementary Fig. 1i-1k).In addition, isoflavone has also been used as an alternative upfront therapy (Zollner et al. Journal of Clinical Medicine. 2021. 10(8):1685).We further tested effects of isoflavone in regulating activation of these kinases.We observed that similar to other chemotherapeutics we tested, isoflavone also induced Akt-pT308 and ERK-p42/p44 in A673 (revised Supplementary Fig. 1l), MHH-ES-1 (revised Supplementary Fig. 1m) and TC-71 cells (revised Supplementary Fig. 1n).Furthermore, combination of isoflavone with SN-38 further promoted activation of both Akt and ERK (revised Supplementary Fig. 1o-1q).Together, these data suggest that chemotherapeutic agents (both alkylator and topoisomerase inhibitors) may generally activate Akt/ERK signaling in Ewing sarcoma cells.1a-c results they use 1 cell line and 24h exposure to SN38 to identify their adoptive mechanisms of resistance, when at this time period it is difficult to conclude that these are actually mechanisms of resistance.There was no real proof that these are mechanisms associated with inhibiting apoptosis/necrosis in the models.Ewing sarcoma is known to be chemo sensitive and presuming that these treatments at 24 hours are leading to chemoresistance is not well justified.

Response:
We thank the reviewer for raising this excellent question.To answer this question, we have performed additional experiments as listed below.6f).These data suggest that increased Akt/ERK activation upon chemotherapy in Ewing sarcoma partially contributes to resistance to 24-hr chemo-treatment.2. In addition, to further test if JAK/GAS6/TAM signaling plays roles in Ewing sarcoma chemoresistance, we have isolated SN-38 resistance single clones from MHH-ES-1 cells following a 2-month selection with ½ IC50 SN-38 treatment.We found these resistant clones were indeed displayed significantly increased resistance to SN-38 treatment in vitro (Fig. R1a), and increased Akt-pT308 and ERK-p42/p44 were observed in these resistant clones (Fig. R1b).Importantly, these SN-38 resistant MHH-ES-1 single clones were more sensitive to filgotinib treatment (JAK1 inhibitor) (Fig. R1c).These data suggest that at least some SN-38 resistant MHH-ES-1 cells display increased Akt/ERK signaling and are sensitized by our identified JAK/GAS6/TAM inhibition.
We are of the opinion that chemo-induced Akt and ERK activation serves as an early signaling event to protect cells from cell death, allowing cells to developing resistance such as long-lasting and stable genetic changes to confer to chemoresistance.We have also included more statements in the revised discussion section to emphasize this notion.

Technical comments:
3. Figure 1: Explain the rationale for the concentrations used in Figure 1 (some seem quite high) as well as the timepoint for analysis at 24hrs.What happens if assess phospho-proteins at 12 hours or 48 hours?Would also be appropriate to measure timeline of drug treatment in the text.
Response: We thank the reviewer for raising this question.From the literature, given it is difficult to determine the comparable doses of chemotherapeutics to clinical doses, different doses of these chemotherapeutic agents have been used in Ewing sarcoma cell line studies.For SN-38, 1 uM concentration is commonly used in previous Ewing sarcoma studies in either Ewing sarcoma cell lines (Grohar et al. Clinical Cancer Research. 2013. 20(5): 1190-1203) or Ewing sarcoma PDX cells (Castillo-Ecija et al. Journal of Controlled Release. 2020. 324: 440-449).For etoposide, 10 uM treatment has been used (Luo et al. Oncogene. 2009. 26(46): 4126-4132) as well as 5 uM (Boehme et al. International Journal of Oncology. 2016. 49(5): 2135-2146).For doxorubicin, up to 10 uM has been used (Luo et al. Oncogene. 2009. 26(46): 4126-4132), and for TMZ, up to 300 uM has been used (Song et al. Scientific Reports. 2016. 6: 22762).We tried a series of doses of each chemotherapeutic agents and presented the data in the revised Fig. 1f-1l and revised Supplementary Fig. 1).
We fully agree with the reviewer that both doses and treatment periods for chemotherapeutics are critical for their effects on cell signaling and cell viability.In the original Supplementary Fig. 1c and 1d, we have included data measuring shorter periods of SN-38 treatment on both A673 and MHH-ES-1 cells and found increased Akt/ERK signaling could be observed as early as 2 hrs post-SN-38 treatment.Following the reviewer's suggestion, we included newly obtained experimental data showing that SN-38 and TMZ increased Akt and ERK activities at 12, 24 and 48 hrs respectively (revised Supplementary Fig. 1e, 1f).Interestingly, SN-38 continued to increase pERK signals with treatment time, however, Akt-pT308 signals peaked at 24 hrs.This was one of the reasons for our analyses on effects of chemotherapeutics on signaling performed at 24 hr post-treatment.

Did the authors assess any combinations tried to see if effects p-status of pathways and try to recapitulate the patient treatment regimen.
Response: We fully agree with the reviewer that it is critical to test if combo-chemo behaves similar to single chemo in our signaling studies.We compared the combination of SN-38 with TMZ to each single treatment in three Ewing sarcoma cells at doses that neither of the dose killed cells at the 24-hr period, and found that chemocombination treatment further increased ERK-p42/p44 with minimal effects on Akt-pT308 compared with each single chemo-treatment in three Ewing sarcoma cells including A673, MHH-ES-1 and TC-71 (revised Supplementary Fig. 1i-1k).In addition, isoflavone has also been used as an alternative for upfront therapy (Zollner et al.Journal of Clinical Medicine.2021.10(8):1685).We further tested effects of isoflavone in regulating activation of these kinases.We observed that similar to other chemotherapeutics we tested, isoflavone also induced Akt-pT308 and ERK-p42/p44 in A673 (revised Supplementary Fig. 1l), MHH-ES-1 (revised Supplementary Fig. 1m) and TC-71 cells (revised Supplementary Fig. 1n).In addition, combination of isoflavone with SN-38 further promoted activation of both Akt and ERK (revised Supplementary Fig. 1o-1q).Together, these data suggest that chemotherapeutic agents (both alkylator and topoisomerase inhibitors) can generally activate Akt/ERK signaling in Ewing sarcoma cells.

6.
Were any upstream kinases, such as AXL, MET on the phosphoprofile?How come activation of these signaling components not noted?Please comment/address.

Response:
We thank the reviewer for raising this great question.We used the Proteome Profiler Human Phopshokinase Array Kit (Biotechne # ARY003C), which includes 37 kinases in total.This panel does not include AXL, MET, MERTK, nor TYRO3.That is why these kinases were not identified through this profiling.We have included a new method section to describe the procedure and reagents for this phospho-profiling in the revised manuscript.

Fig 1m: Status of p-STAT6 and Gas6 expression in A673 xenografts?
Response: We thank the reviewer for this great question.We have performed IHC analyses on isolated tumors from this experiment and observed increased ERK-p42/p44 and pSTAT6 signals in irinotecan treated tumors compared with vehicle control treated tumors (revised Supplementary Fig. 2e).We also tried GAS6 staining in this IHC analysis, but we tried various GAS6 antibodies and failed to find any signals.Since GAS6 is a secreted protein, we thought most secreted GAS6 proteins were lost during tumor fixation steps which yielded low signals.
In addition, we also performed TMZ treatment on xenografted A673 tumors, and found from IHC analysis that increased ERK-p42/p44 and STAT6-pY641 were also increased in TMZ treated tumors (revised Supplementary Fig. 2b).

Show tumor response?
This still does not prove these are associated with driving resistance.Also, the authors should assess with multiple models and chemotherapies…not just irinotecan.For Fig 1m, they are biased towards looking at p-ERK and p-AKT.Should do phosphoarray analysis on these tumors?Other pathways that might be contributing to the "chemosresistance", which again needs to be more convincingly demonstrated.
Response: We thank the reviewer for raising these questions.Following the reviewer's suggestions, we have also performed A673 xenograft experiment treated with TMZ in addition to irinotecan.As shown in the revised Fig. 1m-1o, TMZ treatment reduced tumor growth in nude mice with minimal effects on animal weights (revised Supplementary Fig. 2a).Analyses of isolated tumors showed that compared with vehicle control, TMZ treatment also increased tumor Akt-pT308 and ERK-p42/p44 signals (revised Fig. 1p), which was further confirmed by tumor IHC analysis (revised Supplementary Fig. 2b).These newly included data suggest chemotherapeutic treatment by either irinotecan or TMZ caused activation of Akt and ERK in tumors.We fully agree with the reviewer that although these chemotherapeutic agents induced activation of Akt/ERK in these tumors, it does not necessarily mean activated Akt/ERK signaling contributes to chemoresistance.Our goal in this study is to improve chemotherapy efficacy instead of overcoming chemoresistance.We are of the opinion that Ewing sarcoma tumors may acutely increase Akt/ERK signaling upon chemotherapy to provide a cell survival protection, a period allowing Ewing sarcoma tumors to develop stable resistance mechanisms such as genetic changes.Thus, if we shorten this protection window by inhibiting JAK/TAM/GAS6 signaling as we performed in this study, this would improve chemotherapy efficacy.We have also further clarified this statement in the discussion section in the revised manuscript.
In addition, to further test if JAK/GAS6/TAM signaling plays roles in Ewing sarcoma chemoresistance, we have isolated SN-38 resistance single clones from MHH-ES-1 cells following a 2-month selection with ½ IC50 SN-38 treatment.We found these resistant clones were indeed displayed significantly increased resistance to SN-38 treatment in vitro (Fig. R1a), and increased Akt-pT308 and ERK-p42/p44 were observed in these resistant clones (Fig. R1b).Importantly, these SN-38 resistant MHH-ES-1 single clones were more sensitive to filgotinib treatment (JAK1 inhibitor) (Fig. R1c).These data suggest that at least some SN-38 resistant MHH-ES-1 cells display increased Akt/ERK signaling and are sensitized by our identified JAK/GAS6/TAM inhibition.
Moreover, we fully agree with the reviewer it is a great idea to perform phospho-profiling using xenografted tumors treated with chemotherapy than using Ewing sarcoma cell lines.We have tired this assay using tumor lysates previously but failed.One major technical reason is because the tumors from mice are with quite many blood or blood vesicles, which generates a quite high background noise signals for high-sensitive assays like phospho-profiling.The advantage for using cell lines is that the population of cells are more unified with less heterogeneity and without contaminations from blood.Another reason is that from tumor harvest to obtaining tumor lysates takes more steps and longer time than simply harvesting cultured Ewing sarcoma cells on cell culture dishes.This prolonged processing steps and time would in theory affect signaling dynamics.Thus, supporting these synergy plots in the revised Supplementary Fig. 5a.
In addition, we have further strengthened our conclusion by delaying the treatment till xenografted tumors became larger.In the revised Supplementary Fig. 9b-9e, we xenografted MHH-ES-1 cells to nude mice and didn't start the treatment until tumors are at least 250 mm 3 .Similar to starting treatment earlier (revised Fig. 8l-8o), we observed that even for large tumors that more faithfully mimic clinical settings, compared with single treatment by either irinotecan or MRX-2843, the combination significantly improved the treatment efficacy in suppressing tumor growth (revised Supplementary Fig. 10b-10d).Importantly, no toxicity was observed associated with these treatments, either for early treatments (revised Fig. 8o) or late treatments (revised Supplementary Fig. 10e).
We fully agree with the reviewer that PDX is a critical and more clinically related model to test the synergistic effects of our proposed combination therapy.Following the reviewer's suggestion, we have tried hard to establish Ewing sarcoma PDX models in our lab.We have obtained various Ewing sarcoma PDX tumors from Dr. Peter J. Houghton at UT Health San Antonio, including EW-5, NCH-EWS-1, UTH0584.000,UTHo589.000commonly used in testing effects of various treatments on Ewing sarcoma tumor growth (Robles, et al. Clin Cancer Res. 2020. 26: 3012-3023).We thawed all tumors and isolated single cells for injection into nude mice to obtain fresh Ewing sarcoma PDX tumors.We were only able to recover the PDX tumor growth from NCH-EWS-1 PDX tumor cells.After obtaining fresh NCH-EWS-1 PDX tumors from nude mice, we further collected these tumors for single cell isolation.Afterwards, we injected single cell suspensions of NCH-EWS-1 PDX tumor cells with Matrigel into nude mice subcutaneously.When tumors are observable, we started treatment by vehicle, irinotecan (10 mg/kg, IP), MRX-2843 (10 mg/kg, IP) and the combination.Tumor growth was monitored by caliper measurements (revised Supplementary Fig. 10f).27-days post-PDX cell injection, tumors were harvested and we found irinotecan significantly reduced NCH-EWS-1 PDX tumor growth, while the combination further reduced the PDX tumor growth (revised Supplementary Fig. 10f-10h) with minimal effects on animal weights (revised Supplementary Fig. 10i).These data suggest our proposed combination therapies demonstrate an efficacy in reducing Ewing sarcoma tumor growth in animal models derived from either Ewing sarcoma cell lines or PDX tumor cells.
14. Show that GAS6 is a direct target of STAT6?Or an indirect mechanism?Need to further prove or discuss.
Response: We thank the reviewer for raising this critical question.To test if STAT6 binds directly to GAS6 promoter, first of all, we bioinformatically analyzed if there are potential consensus STAT6 binding sites in 5′ regulatory region of GAS6 gene using Eukaryotic promoter database (SIB, Switzerland).4 potential STAT6 binding motifs are predicted within -2kb region with a p-value smaller than 0.001, including SB1(-353), SB2(-487), SB3(-1440) and SB4(-1706).We then performed cut&run analyses using STAT6 antibody with MHH-ES-1 cells treated with DMSO or SN-38 for 24 hrs.STAT6 immunoprecipitated DNA fragments were analyzed by PCR.As indicated in the revised Fig. 5f, STAT6 demonstrated stronger binding capacity to SB1 and SB3 in MHH-ES-1 cells.Further SN-38 treatment significantly increased STAT6 occupancy on SB1 and SB3 (revised Fig. 5f).These data strongly suggest that STAT6 directly binds SB1 and SB3 in GAS6 promoter, and SN-38 treatment enhances STAT6 localizing to SB1 and SB3, presumably through JAK1-mediated STAT6 phosphorylation.

Fig 3i: Check tumors treated with just irinotecan only for evidence of activation of these pathways/mechanisms of action..do you see evidence of the STAT6-GAS6-TAMK activation Compared to Fig 1m (where not clear of the tumor size or response to Irinotecan) the investigator should do analysis of tumors post-treatment---assess effects on small molecule targets?
Response: We thank the reviewer for bringing up this great question.Following the reviewer's suggestion, we have performed IHC analyses on isolated tumors treated with either irinotecan, UNC2025A or both.Consistent with revised Supplementary Fig. 2e, we observed increased ERK-p42/p44 and pSTAT6 signals in irinotecan treated tumors compared with vehicle control (revised Supplementary Fig. 5p).Moreover, UNC2025A treatment efficiently reduced both ERK-p42/p44 and STAT6-pY641 signals induced by irinotecan treatment (revised Supplementary Fig. 5p).We also tried GAS6 staining in this IHC analysis, but we tried various GAS6 antibodies and failed to find any signals.Since Gas6 is a secreted protein, we thought most secreted GAS6 proteins were lost during tumor fixation steps which yielded low signals.Nonetheless, these newly obtained IHC data support the notion that chemotherapy-induced JAK1/GAS6/TAM signaling may contribute to Ewing sarcoma tumor growth and UNC2025A reduces chemotherapy-induced JAK1/GAS6/TAM signaling to suppress Ewing sarcoma tumor growth.

Any evidence of these being present in relapsed patient tumors?
Response: We thank the reviewer for raising this excellent question.We would love to analyze if compared with primary tumors, relapsed Ewing sarcoma tumors display increased activation of the JAK/STAT6/GAS6/TAM signaling.However, given Ewing sarcoma is a rare pediatric cancer with ~200 cases each year in the US, and usually it is difficult to collect matched primary and relapsed tumors, and even it is difficult to distinguish between treatment naïve or resistant tumors, there is not enough clinical resources for this proposed direction.To date, there has been any noticeable databases for profiling protein changes associated with chemotherapy resistance in Ewing sarcoma.
Although various Ewing sarcoma cell lines at different stages of therapy were isolated including CHLA-9 at diagnosis, CHLA-10/25 after chemotherapy with progressive disease, and CHLA-258 post myeloablative therapy and autologous bone marrow transplantation, these lines from different disease/treatment stages didn't correlate with sensitivity to chemotherapies tested (May et al.PLOS ONE.2013.0080060), maybe because these cell lines were isolated from different patients with distinct genetic background.This suggests these established in vitro cell lines may not be faithful sources for studying chemoresistance.Thus, although this is an appealing and important question, due to the rareness of the disease and lack of clinical resources, we haven't been able to examine if any of our proposed signaling changes are also observed in relapsed, chemo-resistant Ewing sarcoma patients.We acknowledge this is a very important question to address and do hope more clinical resources will be available for Ewing sarcoma research in the future.

Fig 5: Seem like very high doses of SN-38 needed for effects on cell viability...see the effects of phosphorylation at low [ ],
but not on viability when KO STAT6.What are the effects of phosphorylation at these higher doses of chemo?IC50s are 10fold higher than used for WBs.At these higher concentrations (1 0-300uM) what other signaling pathways are being altered?This needs to be discussed as well.
Response: We thank the reviewer for bringing up this question.We used relatively higher doses of SN-38 in cell viability assay in Fig. 5 because our cell viability assays were performed 48 hrs post-treatments.If we evaluate cell viability in a longer period (eg. a week) we can dramatically reduce the SN-38 doses to observe cell death.Following the reviewer's suggestion, we have performed western blotting analyses on the phosphorylation changes at these higher doses of chemo with a 24-hr treatment period.As shown in the revised Supplementary Fig. 7b, we observed that under these conditions, STAT6 depletion similarly reduced SN-38 induced Akt-pT308 and ERK-p42/p44 signals.In addition to the Akt/ERK signaling, we also examined Hippo signaling (Ahmed et al. J Cancer. 2015. 6(10): 1005-1010) and GSK3b (Abe et al.J cancer Metastasis Treat.2020.6:51) phosphorylation that have been reported to be associated with chemotherapy treatment in Ewing sarcoma.To this end, we didn't find significant changes of these phosphorylation events either induced by SN-38 or regulated by STAT6 depletion (revised Supplementary Fig. 7b).These data suggest SN-38 or STAT6 is more likely to regulate specific protein phosphorylation events instead of globally changes on phospho-proteome.We acknowledge that more through phospho-profiling will answer this question, but it warrants a separate study.We have also included more statements in the discussion section for clarification.

Fig 8.
In vivo studies would benefit from assessing the effects on more than 1 cell line model.

Response:
We fully agree with the reviewer and following the reviewer's suggestion, we have also examined the efficacy of this combination therapy using Ewing sarcoma PDX murine models.PDX is a critical and more clinically related model to test the synergistic effects of our proposed combination therapy.Following the reviewer's suggestion, we have tried hard to establish Ewing sarcoma PDX models in our lab.We have obtained various Ewing sarcoma PDX tumors from Dr. Peter J. Houghton at UT Health San Antonio, including EW-5, NCH-EWS-1, UTH0584.000,UTHo589.000commonly used in testing effects of various treatments on Ewing sarcoma tumor growth (Robles, et al. Clin Cancer Res. 2020. 26: 3012-3023).We thawed all tumors and isolated single cells for injection into nude mice to obtain fresh Ewing sarcoma PDX tumors.We were only able to recover the PDX tumor growth from NCH-EWS-1 PDX tumor cells.After obtaining fresh NCH-EWS-1 PDX tumors from nude mice, we further collected these tumors for single cell isolation.Afterwards, we injected single cell suspensions of NCH-EWS-1 PDX tumor cells with Matrigel into nude mice subcutaneously.When tumors are observable, we started treatment by vehicle, irinotecan (10 mg/kg, IP), MRX-2843 (10 mg/kg, IP) and the combination.Tumor growth was monitored by caliper measurements (revised Supplementary Fig. 10f).27-days post-PDX cell injection, tumors were harvested and we found irinotecan significantly reduced NCH-EWS-1 PDX tumor growth, while the combination further reduced the PDX tumor growth (revised Supplementary Fig. 10f-10h) with minimal effects on animal weights (revised Supplementary Fig. 10i).These data suggest our proposed combination therapies demonstrate an efficacy in reducing Ewing sarcoma tumor growth in animal models derived from either Ewing sarcoma cell lines or PDX tumor cells.and pT308.Thus, in this study, we rely on Akt-pT308 as an Akt activation marker but we think Akt-pS473 behave similarly.
3. The activation of JAK1 by chemotherapy needs to be clarified.The text presents the concept that DNA damage response kinases (ATM, ATR, DNAPK) can phosphorylate JAK1 at S571 and this is repeated in the discussion.However, the authors do not mention that a SQ sequence is indicative of an ATM/ATR substrate site.It will be confusing for readers to just jump to JAK1-pSQ terminology.As written there are sections of the results that read as if JAK1 activation occurs in the absence of DNA damage response.Please clarify.
Response: We thank the reviewer for bringing up this critical question and fully agree with the reviewer that we should introduce SQ/TQ phosphorylation by DNA damaging kinases prior to presenting JAK1-pSQ data.In the revised manuscript, we have included background introductions for these DNA damage kinases and pSQ/pTQ phosphorylation motifs.In addition, we have also clarified that JAK1-pSQ requires DNA damage conditions in the revised manuscript.

Does activation of JAK1 by chemo occur in the nucleus or cytoplasm?
Response: We thank the reviewer for this great question.We performed new immunofluorescence microscope analyses on control or SN-38 treated MHH-ES-1 cells and found although SN-38 potently promoted JAK1-pSQ in cells (revised Fig. 7d-7e), it didn't significantly affect JAK1 cellular localization (revised Supplementary Fig. 9h).In addition, we also included JAK1-S571A mutant as a negative control, and observed SN-38 also didn't regulate cellular localization of this phosphorylation-deficient mutant form of JAK1 revised Supplementary Fig. 9h).These data suggest that chemo-induced JAK1 activation didn't regulate JAK1 cellular localization.
To directly examine where chemo-induced JAK1-pSQ occurs, we performed cell fractionation assays and observed that SN-38-induced JAK1-pSQ largely occurred in the nucleus (revised Supplementary Fig. 9g).

5.
The in vivo efficacy studies are promising.I strongly suggest/recommend repeating in vivo studies testing the best combo therapy vs chemo alone and control (limited groups) in mice with established tumors (>250 mm3) to drive higher impact.
Response: We thank the reviewer for this excellent suggestion and cannot agree more with the reviewer to further test effects of our proposed combination therapy on established large tumors to mimic clinical treatments.Following the reviewer's suggestion, we have xenografted MHH-ES-1 cells into nude mice and didn't start the treatments until tumors reached at least 250 mm 3 .As shown in the revised Supplementary Fig. 10b-10e, compared with each of the single treatment by irinotecan or MRX-2843, the combination of MRX-2843 with irinotecan significantly reduced tumor growth (revised Supplementary Fig. 10b-10d) with no observed toxicity to animals (revised Supplementary Fig. 10e).We fully agree with the reviewer that these newly included data further strengthened our confidence in applying proposed combination therapy in treating Ewing sarcoma in clinical trials.

Minor: 6. In Fig 2 the authors show that KD of AXL blocks chemo-induced AKT-pT308 but cabozantinib, a
reasonably potent inhibitor of AXL, does not.This is somewhat confusing and should be discussed.

Response:
We thank the reviewer for bringing up this question.We apologize for the typo in the original manuscript as AXL depletion didn't block SN-38 induced Akt-pT308 in MHH-ES-1 cells (revised Fig. 2e), nor increased Akt-pT308 by additional chemotherapeutics including doxorubicin (revised Supplementary Fig. 3b), etoposide (revised Supplementary Fig. 3e) nor TMZ (revised Supplementary Fig. 3h).We have corrected this statement in the revised manuscript.3a-h.Just as examples, these could be in the supplement.The heat maps are not intuitive.

I recommend showing a cell viability curve for at least a few of the combination cell viability studies displayed in Fig
to SB1 and SB3, presumably through JAK1-mediated STAT6 phosphorylation.

4) Serine phosphorylation of STATs is important for STAT-mediated gene activation. Several kinases including ERKs are involved in STAT serine phosphorylation. Since chemotherapy was shown to induce ERK activation in Ewing sarcoma cells, does the chemotherapy-induced ERK activation have any effect on STAT serine phosphorylation, if so, does this phenomenon contribute to the chemoresistance in Ewing sarcoma?
Response: We thank the reviewer for raising this critical question.We fully agree with the reviewer that in addition to STAT6-pY641, STAT6 can also be phosphorylated by additional kinases such as TBK1 (Chen et al. Cell. 2011. 147(2): 436-446), ERK (So et al. Mol Immunol. 2007. 44(13): 3416-3426) or JNK (Shirakawa et al. J Biol Chem. 2010. 286(5): 4003-4010) on serine residues that differentially regulates its transcriptional activity.Specifically, TBK1 phosphorylates STAT6 at Ser407 to facilitate its Y641 phosphorylation and activation.ERK is critical for IL-induced STAT6 activation.JNK phosphorylates STAT6-Ser707 to suppress STAT6 binding with DNA thus inactivating STAT6.Due to the lack of specific phospho-serine antibodies for ERK-mediated STAT6 phosphorylation, we relied on the pSer-PKC substrate antibody (Cell Signaling Technology #2261) for this test.As shown in the Fig. R2 below, we didn't observe any changes on STAT6-pSer signals.In addition, we found that ERK inhibition by PD0325901 failed to reduce SN-38-induced GAS6 transcription in MHH-ES-1 cells (revised Supplementary Fig. 8f), suggesting ERK and ERK-mediated STAT6 Ser phosphorylation may not play a critical role here.Consistently, PD0325901 didn't affect SN-38 induced STAT6-pY641 signals in MHH-ES-1 cells (revised Supplementary Fig. 8g).These data suggest ERK/STAT6 signaling may not contribute to Ewing sarcoma chemoresistance.patient samples for paired prior-and post-chemotherapy established nation-wide.In addition, given when relapse occurs the recurrent tumors may not be at the same location, whether changes in the recurrent tumors contribute to chemoresistance remains unclear.
Due to the rareness of the disease, prior work in finding Ewing sarcoma chemotherapy resistance mechanisms was initiated from pan-cancer levels or from Ewing sarcoma cell lines.These efforts include data mining of chemoresistance genes from all cancer types followed by validation of Ewing sarcoma related pathways by developing chemo-resistant Ewing sarcoma cell line models (Horbach, et al. 2018. Mol Clin Oncol. 8(6): 719-724); using RNA-Seq to find differential gene expression in CD133 high or low Ewing sarcoma cell lines that establish a correlation of stemness with chemoresistance (Roundhill et al. 2023. Cancers. 15(3): 769) and using Ewing sarcoma PDX models (by establishing three pairs of Ewing sarcoma PDX models from the same patients at primary diagnosis and later stages and focused on CNA (copy number alternations) and SN-38 distributions with no intention to look into chemoresistance profiles (Castillo-Ecija et al. 2020. Journal of Controlled Release. 324 (10): 440-449)).These studies provide further insights into possible chemo-resistant mechanisms; however, clinical validations would be necessary.
Interestingly, a recent study determined the transcriptome changes in 4 Ewing sarcoma patients with good responses to chemotherapy vs. 9 patients with poor responses (Chen et al. 2020. Mol Oncol. 14(5): 1101-1117).Re-analyzing this published data led us to find that increased JAK1 and STAT6 expression were identified in these patients positively correlated with poorer response to chemotherapy (revised Supplementary Fig. 10j).However, due to the small number of patients in this study, we cannot conclude if increased JAK1/STAT6 expression contributes to Ewing sarcoma chemoresistance.
In addition, we are of the opinion that our proposed chemotherapy/JAK1/STAT6/GAS6/TAM/Akt/ERK signaling may not directly contribute to chemoresistance as a stable deregulated signaling, rather activated Akt/ERK signaling will provide a protection for Ewing sarcoma cells to develop genetic changes to confer stable/acquired resistance to chemotherapy.Otherwise, cells will be killed by chemotherapy even before acquired resistance can be developed.We have also included these statements in the discussion section (page 23 line 532).
We hope the reviewer agrees with us that due to the lack of clinical resources for this rare disease, and our proposed signaling may not directly play roles in either intrinsic or acquired chemoresistance in Ewing sarcoma, if deregulation of proposed chemotherapy/JAK1/STAT6/GAS6/TAM/AKT/ERK signaling is observed in relapsed or chemo-resistant Ewing sarcoma patients warrants further investigations in a separate study.

Fig 1m :
Fig 1m: Status of p-STAT6 and Gas6 expression in A673 xenografts?Show tumor response?This still does not prove these are associated with driving resistance.Also, the authors should assess with multiple models and chemotherapies…not just irinotecan.For Fig 1m, they are biased towards looking at p-ERK and p-AKT.Should do phosphoarray analysis on these tumors?Other pathways that might be contributing to the "chemosresistance", which again needs to be more convincingly demonstrated.
Fig 1 to claim chemoresistance without really proving that the cells are chemoresistant.

Fig
Fig 2d-f: Tried only SN-38 on MERTK/AXL depleted cells.They should assess response to other chemotherapies (noted in Figure 1) and additional cell line models.

Fig 3i :
Fig 3i: Check tumors treated with just irinotecan only for evidence of activation of these pathways/mechanisms of action..do you see evidence of the STAT6-GAS6-TAMK activation showing a cell viability curve for at least a few of the combination cell viability studies displayed in Fig3a-h.Just as examples, these could be in the supplement.The heat maps are not intuitive.3.I would avoid the use of JAK1-KD terminology.'KD' is commonly used for knockdown or kinase dead, which can easily lead to reader confusion.4. Fig 6c is unconvincing as displayed.Either replace, move to supplement or delete.The cell fractionation is sufficient to demonstrate nuclear localization.Suggested edits: Line 237 -change 'BCA' to 'TCA' Line 260 -change 'macrophage' to 'macrophages' 17. Fig. 6f-k -These experiments should include a lane that shows the results in cells treated with the different agents alone.They may have effects of baseline signal activation levels which should not be ignored, especially as W blotting captures relative signal levels rather than absolute.Fig 6i-6k: 4 lanes including inhibitor treatment alone.

Figure R1 .
Figure R1.Isolation and characterization of SN-38 resistant MHH-ES-1 single clones.(a) MTT cell viability analyses showing isolated SN-38 resistant SR1-SR4 clones are resistant to SN-38 treatment in vitro.(b) IB analyses of WCL from indicated MHH-ES-1 cells showing increased Akt and ERK activities are observed in SR cells.(c) MTT cell viability assays showing that MHH-ES-1 SR cells are sensitive to filgotinib treatments.

Figure
Figure R2.IB analyses of HA-IPs and WCL from MHH-ES-1 cells stably expressing HA-STAT6 by lenti-viral infection.